The LHD has a long history of investigating patients with abnormalities of phagocytic cell function. These studies include the delineation of clinical, functional, and molecular defects of patients with neutrophil specific granule deficiency, chronic granulomatous disease (CGD), leukocyte adhesion deficiency (LAD), the syndrome of hyperimmunoglobulinE and recurrent infections (Jobs Syndrome) and IRAK4 deficiency. Large cohorts of these patients have been recruited over the years and represent a unique national resource for biomedical research at the NIH. Currently we follow over 150 patients with CGD, about 40 patients with Jobs syndrome, and 30 patients with other phagocyte dysfunction syndromes, including LAD, cyclic neutropenia, neutrophil specific granule deficiency, Chediak-Higashi syndrome, IRAK4 deficiency and NEMO deficiency. We now have EB virus transformed B cells from most of our patients and we have been pleased to share these B cell lines with other intramural or extramural colleagues. We continue to monitor and expand these cohorts of patients who serve as models for long term studies of the clinical consequences of the immune dysfunction in humans. In 2010 we continued our studies of the importance of lactoferrin and other iron chelators in protecting against Aspergillus fumigatus infection, the most common infectious cause of mortality in CGD patients today. We found that while CGD PMN are unable to kill Aspergillus hyphae, their ability to arrest the growth of conidia was identical to that of normal PMN showing a role for nonoxidative mechanisms in host defenses against this organism. We then showed that the neutrophil secretory product, lactoferrin, inhibits conidia germination by sequestering iron, a critical growth factor. Our in vitro studies of the growth inhibitory properties of iron chelating drugs against Aspergillus fumigatus, alone and in combination with first line antibiotics such as amphotericin B, itraconazole, and fluconazole demonstrated antifungal synergy in some combinations. We are currently collaborating with with June Kwon-Chung (LCID) to test whether these drugs work in vivo in aspergillosis in immunosuppressed or CGD mice. (Kol Zarember, 20% effort). In 2010 we completed our gene sequencing data and phenotype correlates in 287 patients with chronic granulomatous disease (CGD) representing 244 distinct families, a cohort large enough to provide sufficent statistical power to compare specific CGD subtypes. The data indicate that survival in CGD patients was highly associated with residual reactive oxygen intermediate (ROI) production as a continuous variable, independent of the specific gene affected. Patients with mutations in p47phox and most patients with missense mutations in gp91phox (excepting mutations of the nucleotide binding and the heme binding domains) had more residual ROI production than patients with nonsense, frameshift, splice or deletion mutations in gp91phox. After adolescence, mortality curves diverged based on residual ROI production. These data have important implications for the management of patients with CGD. Although our data indicate that residual ROI production predicts mortality in CGD, genetic analysis can, in many cases, predict ROI production. In our studies of 273 fully genotyped patients 154 mutations were found, including 55 of which have not been reported previously with 45 in gp91phox, 3 in p22phox and 7 in p67phox. Missense and frameshift mutations in gp91phox were randomly distributed throughout the gene and were generally family specific. Little residual ROI production was observed with nonsense, frameshift, splice or deletion mutations in gp91phox. Unexpectedly, patients with missense mutations affecting amino acids 1-309 (excepting heme-binding His222) exhibited even higher residual ROI production than was observed in patients with p47phox CGD. ROI production was not correlated with protein expression. Mutations in FAD and NADPH binding domains of gp91phox may allow normal protein expression but little residual ROI production demonstrating and confirming the critical role of these domains. While protein detection provides important clues to the diagnosis of CGD, quantitiative measure of ROI, which correlates closely with specific genetic mutations, is more useful for determination of a patients long-term risk and thereby guides therapeutic management. Over the past year, the laboratory has expanded its study of Granulibacter bethesdensis, a recently described bacterial pathogen of CGD patients. Granulibacter is remarkably hypostimulatory of the innate immune system, both in terms of weak activation of the NADPH oxidase and poor stimulation of cytokine secretion. We are working to purify and characterize what appears to be an atypical lipolysaccharide from Granulibacter that activates the limulus LPS test but fails to activate human PMN in keeping with our findings that this microbe may avoid host defenses by using molecular stealth. Biochemical analysis has indicated LPS from G. bethesdensis has an unusual KDO core with an aytpical sugar residue. We have also found that G. bethesdensis is remarkably resistant to complement and antimicrobial peptides. Further studies examining the transcriptional responses of the pathogen to attack by the host and of host cells in response to the pathogen are underway. (Zarember 50% effort, Rogge 50% effort). In 2010 we studied the role of tryptophan metabolism in CGD. Mouse CGD models were recently implicated to have defective tryptophan catabolism as a major regulator of inflammation in CGD. We attempted to validate these findings in human clinical samples and discovered that unlike CGD mice, human CGD patients do not display this defect in tryptophan catabolism. (Zarember, 10% effort). In addition to these projects, in 2010 we have initiated a clinical protocol that will enroll patients to study the development of atherosclerotic disease in patients with immune system disorders. Atherosclerosis, the major cause of heart disease, is thought to relate to dysregulated inflammation in the cardiac blood vessels and possibly results from over production of reactive oxygen species (ROS). We hypothesize that CGD patients, who have deficient production of reactive oxygen species by their phagocytes, may be protected from developing atherosclerosis. The primary endpoint of this study is to determine the prevalence of atherosclerosis in these and other patients with in-born disorders of immune function. The primary endpoint will be assessed using imaging techniques to measure coronary artery calcium scores and the presence or absence of soft plaque. Secondary endpoints include physiologic markers such as blood pressure as well as circulating biomakers associated with heart disease. (Soule 25 % effort). In 2010 we have also iniated another clinical protocol to assess the biochemical responses to interferon gamma in subsets of CGD patients differing with underlying mutation and/or residual NADPH oxidase activity. Interferon gammas has been used clinically in CGD patients to reduce the rates of infection. However, neither the mechanism of its actions nor the wide variation in clinical response among CGD patients is known. This study aims to uncover the biochemical basis of interferons actions in different patient groups.